Population genetic structure of tropical bed bug (Hemiptera: Cimicidae) populations and their breeding pattern in Iraq

Abstract A study was conducted to investigate the population genetic structure and breeding pattern of 140 tropical bed bugs, Cimex hemipterus (F.) (Hemiptera: Cimicidae), collected from 14 infested sites in major cities in Iraq. The samples were genotyped using a set of 7 polymorphic microsatellite markers. High genetic variety was seen among populations, with an average of 2–9 alleles per locus. The number of alleles across 7 microsatellite loci was between 6 and 18. There was a notable disparity in the alleles per loci when comparing the overall population to those within it. The overall population exhibited an average observed heterozygosity of 0.175 and an average expected heterozygosity of 0.730. Among the population, the average observed heterozygosity was 0.173, while the average expected heterozygosity was 0.673. Analysis of molecular variance (AMOVA) revealed that 93% of the genetic variability was within the populations, and 7% was among them. The genetic differentiation coefficient (FST = 0.045), indicates a low degree of genetic differentiation and a high degree of inbreeding (FIS = 0.761), as indicated by notably significant positive inbreeding coefficients. Admixed individuals were revealed using STRUCTURE and neighbor-joining phylogenetic trees, demonstrating moderate gene flow between populations and a lack of genetic structure in the regional groups. Thus, both active dispersion and human-mediated dispersion possess the potential to influence the low population genetic structure of tropical bed bug C. hemipterus populations in Iraq, which can have implications toward tropical bed bug and management strategies.


Introduction
Cimex hemipterus, also known as tropical bed bugs, is an ectoparasite that primarily feeds mostly on human blood during the night.This species has emerged as a noteworthy global public health concern in recent times (Akhoundi et al. 2023).Several factors, such as insecticide resistance, increasing international travelers, trading secondhand items, and ineffective control strategies, may contribute to the global resurgence of bed bugs (Cooper et al. 2015, Raab et al. 2016, Alizadeh et al. 2019).
Bed bug eradication is costly as a result of the difficulties in implementing control strategies, which facilitate the extensive dissemination of bed bugs globally (Akhoundi et al. 2016).At the local scale, the bed bugs associated with humans are highly dependent on the mobility patterns exhibited by human hosts.They may use household items such as furniture, clothing, soiled bedding, bags, and other household items for makeshift conveyances.Bed bugs can also spread in hotel or apartment building rooms by using cracks in walls or gaps around plumbing or ventilation systems (Wang et al. 2010, Booth et al. 2012).Tropical bed bug C. hemipterus biology, including infestation dynamics and prevalence, remains unexplored in Iraq.As bed bugs continue to expand globally, the impact of active dispersal compared to passive dispersal (e.g., human-mediated) on the genetic structure of tropical bed bug C. hemipterus populations in Iraq is still unknown.Consequently, it is becoming increasingly necessary to gain a comprehensive understanding of the basic biology within these populations, and this knowledge will form the essential basis for designing effective management strategies to eradicate this pest.
The genetic structure of bed bug C. lectularius has been investigated in numerous studies (Booth et al. 2012, Saenz et al. 2012, Fountain et al. 2014, Akhoundi et al. 2015, Narain et al. 2015, Raab et al. 2016).However, limited molecular studies have investigated the genetic structure of the tropical bed bug C. hemipterus using microsatellite markers (Seri Masran andAb Majid 2019, Wan Mohammad et al. 2020).
In general, knowledge obtained from microsatellite analysis enables the determination of genetic differences and relatedness within and between populations (Sunnucks 2000, Zhang and Hewitt 2003, Avise 2004, Lee et al. 2008, Gardner et al. 2011, Betson et al. 2014).To date, the genetic structure and breeding patterns of tropical bed bug C. hemipterus have not been studied in Iraq.As an initial step in this endeavor, we used species-specific microsatellite markers to explore the genetic structure of the tropical bed bug C. hemipterus in Iraq.These markers were utilized to assess gene flow and genetic differentiation among the tropical bed bug populations sampled.

Sample Collection
Tropical bed bugs were collected from various infested sites in urban and suburban locations across Iraq between 2020 and 2021 (Fig. 1, Table 1).Genomic DNA was individually extracted from 10 adult specimens randomly selected at each of the 14 infested sites, using the Real Genomic DNA Extraction Kit Mini (RBC Bioscience, Taiwan).The NanoDrop was used to determine the concentration of DNA of each specimen and the DNA purity range of (1.80-2.00) to proceed with PCR amplification using microsatellite markers.
The genetic structure of populations was determined using STRUCTURE version 2.2 (Falush et al. 2003) to group all individuals into clusters displaying their mixing.The program was running with 10 runs for each possible value of K (number of genetic clusters), and with a burn-in period of 100,000 and 100,000 Markov Chain Monte Carlo steps.The value of K was determined to be 15 (Evanno et al. 2005).STRUCTURE HARVESTER was utilized to estimate the most likely number of clusters in the dataset (Earl and VonHoldt 2012).Finally, Cluster Markov Packager across K (CLUMPAK) was used to generate a structure result analysis graphical representation (Kopelman et al. 2015).The GenAlEX v.6.5 program was utilized to assess the genetic differentiation between populations by analysis of molecular variance (AMOVA) (Peakall and Smouse 2012).In addition, Bottleneck v1.2.02 software was used to evaluate the potential for a bottleneck effect (Piry et al. 1999).

Results
A total of 140 individuals from 14 populations were successfully genotyped using 7 microsatellite loci.The electropherogram obtained from fragment analysis showed peaks in all samples.The 7 microsatellite loci exhibited polymorphism in all populations, with alleles ranging from 6 to 18 per locus (Table 3).The mean observed heterozygosity (H obs ) and expected heterozygosity (H exp ) were 0.730 and 0.175, respectively (Table 4).The average H obs was observed to be lower than the average H exp .PIC values greater than 0.5, the mean PIC value was 0.705 (Table 4).
Based on the analysis among 14 populations, the average observed heterozygosity deviated by 0.173 from the expected value of 0.673 (Table 5).The average fixation index inbreeding coefficient (F IS ) for all populations was 0.755.The mean value of genetic differentiation among all populations was low (F ST = 0.045, 95% CI = 0.009-0.079)(Table 5).The inbreeding coefficient F IS value was 0.761 (95% CI = 0.681-0.825).The mean relatedness coefficient of r was found to be 0.051 (95% CI = 0.011-0.087).
The molecular variance analysis (AMOVA) revealed that 93% of the variation was found within populations.Conversely, only 7% of the molecular variance was found among the populations.This result indicated a low genetic differentiation observed in the population of tropical bed bug C. hemipterus, implying low gene flow due to inbreeding (Fig. 2).To estimate the genetic distance, NJ was used.The results obtained from the NJ tree showed that the 14 populations can be grouped into 3 clusters with high bootstrap values (Fig. 3A).Bottleneck analysis assessed recent bottleneck events, which indicated shift in 12 out of 14 populations (Table 6).The evidence of bottleneck events was assessed by excess homozygosity in the populations.This could be due to a decrease in effective population size of tropical bed bug due to control activities and the speed at which the population recovered (Freeland 2005).
The STRUCTURE software was used to analyze the population structure of tropical bed bug C. hemipterus.The high values of LnP (K) and Delta K, were observed when K was assessed as 3 (Mean LnP [K]: -3172.6;Delta K: 36.77);Fig. 3B.The results, as shown in Fig. 3C and described by Evanno et al. (2005) consist of plots illustrating the posterior probabilities for the K = 3 genetic cluster.The NJ tree also revealed the presence of 3 clusters.Eleven populations showed nearly full membership in one of the 3 genetic clusters, whereas the remaining 3 populations showed a mixed membership with 3 genetic clusters (Table 6).

Discussion
The analysis of the population genetics of tropical bed bug C. hemipterus in Iraq based on genotyping 140 individuals from 14 populations using 7 microsatellite loci was successfully applied for analysis of genetic diversity, and the population dynamics of tropical bed bug in Iraq.
The presence of heterozygous alleles in all samples, as evidenced by the peaks observed in the electropherograms, highlights the genetic diversity within the studied populations.The polymorphism exhibited by the 7 microsatellite loci, with alleles ranging from 6 to 18 per locus (Table 3), underscores the variability present across populations.This finding is consistent with previous population genetic investigations conducted on the tropical bed bug C. hemipterus in Malaysia (Seri Masran and Ab Majid 2019, Wan Mohammad et al. 2020).
The mean observed heterozygosity (H obs ) of 0.730 suggests a relatively high level of genetic variation within individuals.In contrast, the expected heterozygosity (H exp ) 0.175 reflects the genetic diversity anticipated under Hardy-Weinberg equilibrium (Table 4).This disparity suggests that the actual genetic diversity within the populations is much higher than what would be expected under Hardy-Weinberg equilibrium.This difference can arise due to several factors that influence population dynamics, like natural selection, genetic drift in smaller populations, or migration of individuals between populations (Gaggiotti et al. 2009).
The mean PIC value of 0.705 (Table 5) indicates that the microsatellite markers used in the study are highly informative and capable of discriminating between different alleles.The average F IS of 0.755 (Table 5) suggests a moderate level of inbreeding within populations.The combination of high PIC values and moderate F IS values suggests that while genetic diversity exists, there is also evidence of some level of inbreeding.This is further supported by the F IS value of 0.761, highlighting the potential mating patterns, and genetic relatedness within populations.The low mean value of genetic differentiation (F ST = 0.045) among populations and the low relatedness coefficient of r (0.051) emphasize that gene flow between populations is significant despite the potential for inbreeding.The heightened likelihood of inbreeding can be attributed to several factors, such as pest control, human-mediated movement, and infrequent additional introduction events per infestation (Booth et al. 2015).
The AMOVA result (Fig. 2) indicated that 93% of the genetic variation exists within populations, while only 7% is attributed to differences between populations.This finding suggests genetic diversity exists within population rather than between different populations.The NJ tree analysis (Fig. 3A) further supports this, with the populations forming 3 clusters with high bootstrap values.The previous study, done by Seri Masran and Ab Majid (2019) regarding the population genetic structure and breeding pattern in Malaysia supports our results.The formation of 3 distinct genetic clusters suggests regional genetic structuring among the C. hemipterus populations in Iraq.The AMOVA result and NJ tree analysis combination suggest that geographic or environmental factors might contribute to the observed genetic structuring.The formation of distinct genetic clusters attribute to specific local conditions.
Using the STRUCTURE software to analyze population structure revealed 3 distinct genetic clusters, as supported by high values of posterior probabilities and Delta K (Fig. 3B and C).Similarly, Narain et al. (2015) study on the bed bug C. lectularius population in the USA reported 3 genetic clusters.This implies the presence of different genetic groups within the sampled populations.In essence, the STRUCTURE analysis reinforces the patterns observed in the AMOVA and NJ analysis.It provides visualization and quantification of the genetic clusters, offering additional support to the idea of the tropical bed bug C. hemipterus population in Iraq exhibiting regional genetic structuring.
Our finding uncovered a mod shift in the 12 out of 14 studied populations of tropical bed bug, indicating a recent bottleneck event.This observation raises the possibility that tropical bed bug populations may have undergone a reduction in effective population size due to eradication efforts, including insecticides or adaption to repeated cycles of inbreeding.Saenz et al. (2012) suggested that it might be possible that Bed bug populations experienced genetic bottleneck events during the control measures such as the use of insecticides.
In conclusion, the tropical bed bug infestation in Iraq can be attributed to low genetic diversity within and between populations.It can be attributed to the mating between more related individuals.In addition, a low genetic diversity suggested 3 genetic clusters found in populations.The passive dispersal and bottleneck events indicate the low level of genetic differentiation observed between

Fig. 1 .
Fig. 1.The sampling locations of tropical bed bug C. hemipterus in Iraq are shown on the map.Table 1 contains abbreviations for collection.

Table 1 .
Details to collecting tropical bed bug samples from sleeping beds in major Iraqi cities

Table 2 .
Characteristics of 7 microsatellites developed for bed bug C. hemipterus

Table 3 .
Details regarding the number of alleles per each population of bed bug C. hemipterus from Iraq for 7 microsatellites loci.

Table 5 .
Information regarding the observed heterozygosity (H obs ), expected heterozygosity (H exp ), and fixation index inbreeding coefficient (F IS ) for each of all the 14 sampled, and summary of F statistics values for 95% confidence intervals grouped as the region and overall population Variation in molecular diversity within and among tropical bed bug C. hemipterus populations in Iraq through the comparable percentages.